The extensive study of diabetes has resulted in insulin being arguably the best understood of all protein molecules. Consequently, insulin has become the preferred substrate to probe the effects of alterations in primary structure on higher orders of protein structure and function. Recombinant DNA technology facilitates the generation of novel insulin analogs for SAR and therapeutic applications. The catalogued effects of these alterations hopefully will unlock the rules governing the relationship between primary and higher orders of protein conformation. However, such modifications in primary structure have been relatively minor in relation to the native sequence. However, such limited diversions from the native sequence provide little insight as to what lies along more divergent pathways.
The pursuit of biochemistry is to design artificial molecules to perform designated functions rather than to rely on the chance discovery of a naturally occurring compound possessing the desired properties. Notwithstanding significant advances, the art is essentially barren of examples of synthetic analogs which differ markedly in primary structure from their naturally occurring counterparts. The instant invention uses the well characterized insulin molecule to embark on the development of a synthetic analog of proinsulin which is markedly different in structure and physical properties from the naturally occurring proinsulin molecule and known proinsulin analogs.
Insulin is a protein consisting of two subunit polypeptides commonly referred to as the A-chain and the B-chain covalently cross linked via disulfide bonds. Human insulin, one representative example of the insulin structure, may be diagrammed as shown in FIG. 29A. The biochemical pathway for the production of insulin is well known in the art and may be found in general references on the subject. (See e.g., Stryer, L., Biochemistry, 2nd. Ed., 1981, W. H. Freeman & Co., San Francisco, pp. 847-848). The naturally occurring in vivo biochemical route to insulin leads through the preproinsulin and proinsulin intermediates.
Insulin is recombinantly produced via the expression of proinsulin followed by enzymatic processing. Proinsulin, the immediate precursor of insulin, is a single chain protein. The two chain insulin molecule is produced by the excision of an internal region, commonly referred to as the C-region or C-peptide, of proinsulin. Subsequent to the formation of the intrachain and inter-chain disulfide cross-linkages, the internal polypeptide sequence (C-peptide) is deleted by the action of the trypsin and carboxypeptidase B enzymes resulting in the functional insulin molecule.
The proinsulin gene is translated in the order corresponding to the B-chain/C-peptide/A-chain amino acid sequence. Since recombinant insulin production begins with proinsulin rather than the preproinsulin molecule, the recombinant proinsulin molecule characteristically possesses a methionine residue at its amino-terminus. Consequently, this met derived from the N-terminus of proinsulin is carried through and remains at the amino terminus of the insulin B-chain. This methionine residue is not intrinsically removed by the bacterial host cell. It is therefore necessary to chemically or enzymatically remove this N-terminal methionine in vitro to achieve the native proinsulin or insulin molecule.
The action of methionyl amino peptidase (MAP), a protein indigenous to E. coli, will remove an N-terminal deformylated methionine provided the second residue is not arginine, aspartate, glutamine, glutamate, isoleucine, leucine, lysine or methionine. Examination of the primary structure of the insulin molecule, human insulin being a representative example shown in FIG. 29, demonstrates that the-N-terminal residue of the B-chain, corresponding to the N-terminal residue of natural proinsulin, is phenyalanine. This transcriptional and translational order prevents the removal of the N-terminal methionine of the recombinantly E. coli produced proinsulin molecule by MAP. However, the N-terminal amino acid of the A-chain is glycine whose presence does not inhibit the action of MAP. Thus, if one could reverse the sequence of translation from B-chain/C-peptide/A-chain to A-chain/C-peptide/B-chain, the intrinsic action of MAP would eliminate the N-terminal methionine. This would consequently obviate the need for post-translational removal of the N-terminal Met thereby incurring a substantial commercial and technical advantage.